PROJECT SUMMARY The goal of this proposal is to determine the rules governing the activation of the receptors in the tumor necrosis factor receptors super family (TNFRSF), for aiding the development of therapeutic antibodies targeting these receptors. There are genuine needs for in-depth understanding of the mechanism by which these receptors are activated, as many of them are targets for antibody-based immunotherapy. While receptor activation accompanying receptor clustering is a general phenomenon observed for receptors of the TNFRSF, existing approaches of developing agonistic/antagonistic antibodies rely largely on trial and error, and no correlation between antibody affinity and functionality has been observed. The proposed research is based on our recent finding that, for a few members of the TNFRSF, the receptor transmembrane helix (TMH) alone can mediate higher-order receptor clustering to drive downstream signaling and that an important role of the receptor ectodomain in the absence of ligand is preventing the TMH-mediated receptor activation. Thus developing antibodies that specifically modulate the auto-inhibitory state of the ectodomain would yield more selective and efficient activators or inhibitors of the receptors. To investigate the general applicability of this mechanism, we will perform structural and functional studies of receptor TMH oligomerization and pre-ligand association for other members of the TNFRSF. Based on our understanding of the mechanism of receptor autoinhibition, we will develop high-throughput technology for discovering antibodies that activate or inhibit members of the TNFRSF in conformation-specific manner. Specifically, in Aim 1, we will perform a thorough survey of receptor TMH clustering to identify TNFRSF members with TMHs that are capable of forming higher- order interaction network and driving signaling. In Aim 2, we will characterize the pre-ligand association structures for a few representative members of the TNFRSF to understand how receptor ectodomain physically inhibit TMH clustering. In Aim 3, we will broadly survey the basic requirements of receptor activation to test our proposed 3-2 rule of receptor activation. In Aim 4, we will exploit the autoinhibition concept to implement a yeast display technology to screen for nanobodies/Fabs that specifically break or stabilize the pre-ligand ectodomain association, as means of activating or inhibiting the receptors, respectively. The proposed research will significantly advance our mechanistic understanding of receptor activation for the TNFRSF while potentially discovering specific agonistic or antagonistic agents for several TNFRSF members such as DR5, TNFR1, TNFR2, OX40, 4-1BB, CD40, and GITR, all of which are important immunotherapy targets.